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Amyloid β-protein fragment 31–35 forms ion channels in membrane patches excised from rat hippocampal neurons
PII: S 0 3 0 6 - 4 5 2 2 ( 0 1 ) 0 0 2 4 4 - 5
Neuroscience Vol. 105, No. 4, pp. 845^852, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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AMYLOID L-PROTEIN FRAGMENT 31^35 FORMS ION CHANNELS IN MEMBRANE PATCHES EXCISED FROM RAT HIPPOCAMPAL NEURONS J.-S. QI and J.-T. QIAO* Department of Neurobiology, Shanxi Medical University, Taiyuan, Shanxi 030001, PR China
AbstractöInside-out membrane patches excised from rat hippocampal neurons were used to test if ion channels could be formed by fragment 31^35 of amyloid L-protein. The results showed: (1) after application of fragment 31^35 of amyloid L-protein (5 WM) to either the inner or outer side of the patches, spontaneous currents could be recorded from those patches that had previously been `silent'; (2) the fragment 31^35-induced conductance was cation-selective with a permeability ratio of PCs /PCl = 23; (3) di¡erent levels of conductance, ranging from 25 to 500 pS, could be recorded in di¡erent patches, and in some cases, di¡erent conductances and spontaneous transitions among them could be recorded in a single patch; and (4) application of ZnCl2 (1 mM) to the inner side of the patches reversibly blocked the newly formed channel activity; a similar e¡ect was observed after application of CdCl2 (1 mM). These results show that fragment 31^35 of amyloid L-protein can insert into membrane patches from both sides and form cation-selective, Zn2 - and Cd2 -sensitive ion channels. It is proposed that fragment 31^35 in amyloid L-protein might be the shortest active sequence known to date to form ion channels across neuronal membranes. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: amyloid L-protein fragment 31^35, inside-out membrane patch, hippocampal neurons, formation of ion channels, Zn2 , Cd2 .
excised from hypothalamic neurons. Simmons and Schneider (1993), using the whole cell voltage clamp technique, have reported that ALP25^35, a short form of ALP fragments which is also neurotoxic, induces a novel ionic conductance in sympathetic ganglion neurons. Furthermore, the observation of Mirzabekov et al. (1994) has shown that ALP25^35 spontaneously incorporates into planar lipid bilayers to form ion-permeable channels. We have demonstrated in a recent study (Yan et al., 1999) that ALP31^35, a shorter fragment of ALP, is able to induce apoptosis in cultured cortical neurons similar to that observed with ALP1^40 or ALP25^35. The neurotoxic action of ALP31^35 has also been shown in cultured basal forebrain GABAergic neurons (Pakaski et al., 1998) and septal cholinergic neurons (Wang et al., 1999). Here, we have investigated whether this shorter fragment of ALP, ALP31^35, can form ion channels across the membrane patch of acutely dissociated hippocampal neurons.
Several lines of evidence have demonstrated that amyloid L-protein (ALP), a main component of the senile plaques in Alzheimer's disease (AD), is neurotoxic and contributes to neuronal dysfunction and death (Pike et al., 1991; Blanchard et al., 1997; Mattson, 1997). The mechanisms of ALP neurotoxicity may involve calcium overloading (Wiss et al., 1994; Korotzer et al., 1995; Ueda et al., 1997; Ma et al., 1998), generation of free radicals (Behl et al., 1994; Manelli and Puttfarcken, 1995; Goodman et al., 1996), modulation of endogenous channels (Etcheberrigaray et al., 1994; Fraser et al., 1997), and/or the formation of new ion channels (Arispe et al., 1993a,b; Mirzabekov et al., 1994; Kawahara et al., 1997; Lin et al., 1999). Among the neurotoxic e¡ects cited above, the formation of new ion channels has provoked extensive interests. Arispe et al. (1993a,b) and Kawahara et al. (1997) have shown that ALP1^40, the commonly observed type of ALP in senile plaques, can form new cation channels in arti¢cial lipid bilayers and in membrane patches
EXPERIMENTAL PROCEDURES
Solutions and isolation of hippocampal neurons
*Corresponding author. Tel.: +86-0351-4135052; fax: +86-03514083014. E-mail address: [email protected] (J.-T. Qiao). Abbreviations : ALP, amyloid L-protein; ALP31^35, fragment 31^35 of amyloid L-protein ; ALP25^35, fragment 25^35 of amyloid Lprotein; ACSF, arti¢cial cerebrospinal £uid; AD, Alzheimer's disease; EGTA, ethylene glycol-bis(L-aminoethyl ether)N,N,NP,NP-tetraacetic acid; Er , reversal potential ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulfonic acid); Po , probability of channel opening; Vp , potential in pipette.
The following solutions (in mM) were used during the course of the dissociation procedure: (1) arti¢cial cerebrospinal £uid (ACSF): NaCl 126, KCl 5, CaCl2 2, MgSO4 2, NaH2 PO4 1.5, NaHCO3 25, glucose 10 (pH 7.4); (2) ACSF with lower concentration of calcium: NaCl 126, KCl 5, CaCl2 0.2, MgSO4 2, NaH2 PO4 1.5, NaHCO3 25, glucose 10 (pH 7.4); and (3) incubation solution: NaCl 130, KCl 5.4, CaCl2 1, MgCl2 1, HEPES 10, glucose 25 (pH 7.4). Symmetrical CsCl solution, that was used as both bath and pipette solution during single channel 845
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recording, possessed the following components (mM): CsCl 140, CaCl2 0.5, MgCl2 0.5, EGTA 1, HEPES 5 (pH 7.4). The experiments were carried out on 28 Wistar rats aged about 20 days (supplied by the Research Animal Center of Shanxi, PR China, with approval of Shanxi Committee on Ethics of Animal Research), and all e¡orts have been made to minimize the number of animals used and their su¡ering. Single neurons were dissociated from the hippocampus as described by Kay and Wong (1986) with minor modi¢cations. In brief, the brain was rapidly removed under the anesthesia with ether and placed in cold (0V3³C) ACSF with a lower concentration of calcium; the hippocampus was dissected out from the cooled brain tissue; slices of 400^600 Wm thickness were cut with a razor blade and transferred into the ACSF bubbled with 95% O2 and 5% CO2 . After at least 1 h, the hippocampal CA1 regions were micro-dissected and cut into small pieces of about 1 mm2 in incubation solution; then the pieces were placed into the incubation solution containing pronase E (1 mg/ml, Merck, Germany) and treated for 30 min at 33³C. After digestion, the pieces were dispersed by trituation with a graded series of Pasteur pipettes. Then, three drops of cell suspension were added to coverslip coated with poly L-lysine (0.1%) to improve cell adherence. After cells settled down on the glass bottom, the incubation solution was replaced with 140 mM CsCl solution (recording solution) before sealing the patch pipette.
ALP channel current recording and single channel analysis Patch clamp experiments were performed following standard procedures (Hamill et al., 1981). Recording pipettes were made on a two-step vertical puller (PP-830, Narishige), and ¢re-polished with a microforge (MF-830, Narishige). The pipette resistance, when ¢lled with 140 mM CsCl solution, was between 8 and V15 M6. Inside-out con¢guration of membrane patch was obtained by rapidly withdrawing the pipette after forming a giga-ohm seal. Channel currents were ¢ltered at 1 or 2 kHz (33 dB), ampli¢ed by a patch clamp ampli¢er (Axopatch 200B, Axon Instruments, USA), and stored on the hard disk of an IBM computer through a 12-bit 333 kHz A/D converter (Digidata 1200B, Axon Instruments, USA). Single channel data acquisition and o¡-line analysis were carried out with PCLAMP 6.04 software (Axon Instruments, USA). Administration of ALP fragments ALP31^35 and ALP25^35 were dissolved in double distilled water as concentrated stock solutions (1 mM) and stored at 320³C. The ¢nal concentration of ALP fragments added to bath solution or pipette solution was 5 WM. Before adding ALP fragments to bath solution (i.e. inner or cytoplasm side of the patch), the excised membrane patch was exposed to sym-
Fig. 1. Representative channel activities induced by adding fragment 31^35 of ALP (5 WM) to the outer side of an inside-out membrane patch. Recordings shown here were obtained 6 min after application of ALP31^35 in symmetrical CsCl solution (140 mM). Vp is indicated above each set of tracings. Channel openings are upward when Vp is negative, or downward when Vp is positive.
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metrical Cs solution for at least 10 min in order to verify that the membrane patch was electrically `silent' by blocking the possibly existed endogenous channels in the patch, as mentioned below. After appearance of ALP channel activity, bath solution was replaced with CsCl solution containing 1 mM ZnCl2 or 1 mM CdCl2 through a gravity-driven multitube perfusion system. The distance from the opening of perfusion tube to the tip of patch pipette was about 100 Wm. In the case of application of ALP fragments to pipette solution (i.e. outer or extracellular side of the patch), the tip of pipette was ¢rst back-¢lled with CsCl solution, then ALP fragments solution was added to the distal end of the CsCl solution in the pipette. Thereby, when it was used for recording, a control period in the absence of ALP was achieved (Kawahara et al., 1997). ALP31^35, ALP25^35, CsCl, ZnCl2 , and CdCl2 were all purchased from Sigma (St. Louis, MO, USA). Experiments were performed at room temperature (22^27³C). The statistical data are expressed as means þ S.D. A P value of = 0.05 was considered statistically signi¢cant.
RESULTS
Endogenous potassium channels in the excised patches were blocked by using a K -free solution containing 140 mM CsCl on both sides of membrane patches. Calcium current was prevented by adding 1 mM EGTA and sodium channels were inactivated by holding the membrane potential between 340 and V+40 mV. To ensure the absence of endogenous channels, recordings were made from a control group with no application of ALP fragments in the bath or pipette solutions. For each patch, an initial control recording was made at a high ampli¢er gain (100 mV/pA) and with di¡erent levels of potential in pipette (Vp , 340V+40 mV) before application of ALP fragments to ensure the absence of endogenous activity. The minimum control time was 10 min or 4 min in experiments for bath or pipette application of ALP fragments, respectively. ALP31^35 forms ion channels across neuronal membrane patches similar to ALP25^35 Under the above conditions, most of the patches that
Fig. 2. Open-time distribution of channel currents induced by fragment 31^35 of ALP. Data were obtained at +40 mV of Vp and symmetric CsCl solution ; ALP31^35 (5 WM) was added to the outer side of the patch. The open-time histogram was best ¢tted by a two-exponential function. The time constants d1 , and d2 are indicated in the graph.
Fig. 3. Di¡erent amplitude of channel currents induced by fragment 31^35 of ALP at the same Vp (340 mV). Symmetric CsCl solutions were used, and ALP31^35 (5 WM) was added to the outer side of the patch. The four groups of recordings (A, B, C and D) came from di¡erent membrane patches. Main conductance values estimated were 34, 55, 82, and 328 pS in A, B, C, and D, respectively. Note that di¡erent levels of conductance were also observed in the same membrane patch (B and C).
were untreated with ALP fragments (10/11) remained electrically silent for up to 60 min. Only 9% (1/11) of these patches displayed occasional current activities. However, channel activities appeared in all patches (100%, 7/7) between 4 and V16 min after exposure of the inner side of the membrane patches to 5 WM ALP25^ 35; 93.5% (14/15) of patches displayed similar channel currents when ALP25^35 was added to the outer side of the membrane via the pipette. A shorter fragment, ALP31^35, composed of only ¢ve amino acids, induced similar channel activities in 92.3% (12/13) or 87.5% (7/8) of membrane patches after the peptide was added into bath or pipette solution, respectively. Fig. 1 shows the single channel activity induced by adding ALP31^35 to the outer side of a patch at di¡erent Vp . The histogram of ALP31^35 current amplitudes could be ¢tted well to a normal distribution (data not shown); the mean open time (Topen ) for the channels was 1.20 þ 0.31 ms (n = 8) at +40 mV of Vp . An open-time histogram was best ¢tted by a two-exponential function (Fig. 2). Characteristics of ALP fragment-induced channel activity Multilevel conductance and spontaneous transition. Unlike the endogenous channels, ALP fragment-induced currents in most of patches showed substantial variability in amplitude. The conductances of the ALP31^35-
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Fig. 4. Spontaneous transition between the di¡erent levels of conductance formed by adding fragment 31^35 of ALP. Graphs A, B, C, and D represent sample currents from four di¡erent membrane patches. Symmetrical CsCl solution was used. Vp was given below in each recording. Numbers on the right side of each recording represent the di¡erent conductance values, and `c' represents closed state of the channels.
induced channels ranged from 25 to 500 pS (n = 19). Fig. 3 illustrates the variability in amplitudes of wellde¢ned channel currents from four di¡erent membrane patches; the main values of conductance from samples A^D, respectively, were 34, 55, 82, and 328 pS at the same Vp (340 mV). In addition, the spontaneous transitions of conductance between di¡erent levels were also found in the same patches, though it occurred in not more than one-¢fth of patches with well-de¢ned channel currents. These spontaneous transitions are illustrated in Fig. 4. Transitions of two sample currents from lower conductance to higher conductance are depicted in A and B, being increased by 80% and 83%, respectively; sample C shows frequent transitions of a channel conductance among di¡erent levels; while the records in D show abrupt changes in conductance from 78 pS to 150 pS and from 115 pS to 195 pS, respectively. The fact that the channel could be closed abruptly and completely from the higher level of conductance excludes the possibility that the existence of a higher level of conductance is owing to the `summation' of conductance from two or more channels. The characteristics of multilevel conductance and spontaneous transition were also observed when ALP25^35 was used (data not shown). Voltage dependence of channel opening. The open frequency of the channels changed with the alteration of transmembrane potential. Topen and the probability of channel opening (Po ) showed similar voltage dependency. For example, when Vp was changed from +20 mV to +40 mV, Topen increased from 0.842 þ 0.27 ms to 1.203 þ 0.31 ms (n = 8, P 6 0.05), and the mean Po from 0.018 þ 0.011 to 0.07 þ 0.05 (n = 6, P 6 0.05). Cation-selectivity of ALP31^35 channel. To identify the ion carrying the current, we observed the channel activities induced by ALP31^35 and measured the shift of the reversal potential (Er ) while changing the [Cs ]i
from 140 mM to 70 mM (n = 14). As expected, the channel current was about 0 pA in symmetrical CsCl solution ([Cs ]i /[Cs ]o = 140/140 mM) at 0 mV of Vp ; by contrast, when asymmetrical CsCl solution ([Cs ]i /[Cs ]o = 70/140 mM) was used, obvious inward currents were invariably observed at Vp = 0 mV. Furthermore, in the experiments for measuring the Er , it was found that Er , being about 0 mV in symmetrical solution, was shifted towards the negative value of Vp in asymmetrical solution, becoming 316.2 mV (Fig. 5). The value of the new Er is close to Cs equilibrium potential (317.8 mV), and it corresponds to a PCs /PCl of 23. This result indicated that under these conditions, the main charge carrier of current is Cs . Blockade of ALP fragment-induced channel activity by Zn2+ and Cd 2+ Representative experiments in Fig. 6 show that addition of ZnCl2 (1 mM) to the bath solution drastically reduced the amplitude and the open frequency of channel currents induced by ALP31^35 (Fig. 6A) or ALP25^ 35 (Fig. 6B) applied through pipette solution. This action seemed to be reversible, in that the channel activity reappeared when Zn2 -containing bath solution was replaced with normal bath solution. We also examined the e¡ect of Cd2 on the channel activity induced by ALP fragments (n = 9). CdCl2 (1 mM) blocked ALP31^35-induced channel currents in 8/9 patches tested (Fig. 7). The Po of the channel in CdCl2 decreased from 0.035 to 0.021, and the average current amplitude from 3.17 þ 0.632 to 1.324 þ 0.38 pA. The suppressive action of Cd2 on ALP31^35 channels was reversible.
DISCUSSION
Since Arispe et al. (1993a,b) found that ALP1^40
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Fig. 5. Channels formed by fragment 31^35 of ALP are cation-selective. (A) Channel activities recorded in symmetrical and asymmetrical CsCl solution. Di¡erent levels of Vp are shown on the left of corresponding recordings. The solid line in each trace represents channel closed state. (B) Amplitudes of ALP31^35 channel currents plotted as a function of Vp . Er was shifted from about 0 mV in control (b) to 316.2 mV after asymmetrical CsCl solution was used (a). PCs /PCl was estimated using the following equation: Er = (RT/F) ln{{PCs [Cs]o +PCl [Cl]i }/{PCs [Cs]i +PCl [Cl]o }}, by inserting the values of concentrations that were presently used ([Cs]o = 140 mM, [Cs]i = 70 mM, [Cl]o = 140 mM, [Cl]i = 70 mM) into the equation, and the result showed: PCs /PCl = 23.
could induce a novel inward ionic conductance in bilayer membranes, the mechanisms by which ALP a¡ects ion £ux across the membrane have been widely studied. The present results demonstrate that not only ALP25^35 but also ALP31^35 can spontaneously insert into membrane patches from both sides and form cation-selective, Zn2 and Cd2 -sensitive ion channels. The characteristics of
ALP31^35 channels are quite similar to that of channels formed by ALP1^40 or ALP25^35 in arti¢cial lipid bilayers (Arispe et al., 1993a,b; Mirzabekov et al., 1994) or neuronal membrane (Simmons and Schneider, 1993; Kawahara et al., 1997) as reported previously. ALP31^35 is the shortest sequence of ALP known to date to form transmembrane channels.
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Fig. 6. Reversible blockade of channels formed by fragments 31^35 and 25^35 of ALP by Zn2 . Membrane patches were exposed to symmetrical CsCl solutions, and ALP31^35 (A) or ALP25^35 (B) was applied from the outer side of the patch. After appearance of channel currents (top), bath solution containing 1 mM of ZnCl2 was added to the inner side of the patches (middle); the channel activities in recovery were recorded 2 min later after replacing Zn2 -containing bath solution with normal bath solution (bottom). Vp was 340 mV during all recordings above.
Formation of ALP31^35 channel An important question is how a small peptide of only ¢ve amino acids might form a membrane-spanning ion channel. It has been reported (Barrow et al., 1992) that ALP and some of its degradation molecules, in aqueous solution, can form stable dimers, trimers and tetramers and, moreover, the rate of aggregation is the fastest for the hydrophobic sequence 29^42 of ALP. In addition, the critical role of isoleucine in 31 and 32 and of methionine in 35 position for the aggregation and toxicity of ALP25^ 35 has been emphasized (Pike et al., 1993). Considering the facts that ALP fragment 31^35 (Ile-Ile-Gly-Leu-Met) is constituted of hydrophobic amino acids and contains all of the three key amino acids responsible for the aggregation and toxicity of ALP25^35, we postulate that this shorter peptide cannot only exhibit the apoptogenic activity on neurons, as proved previously (Yan et al., 1999), but also can easily incorporate itself into the membrane and form special membrane-spanning ionophore structures. As a corollary, if new channels could be formed by the aggregation of ALP31^35 fragments, different polymers incorporated into the cell membrane would be responsible for the di¡erent levels of conductance; the spontaneous transition of conductance states, as observed in the present study, might be the result of the rapid inter-conversion among di¡erent conforma-
tions of the same ALP31^35 polymers. As pointed out by Durell et al. (1994), the inter-conversion does exist among di¡erent subtypes of polymers formed by ALP fragments. Cation-selectivity of ALP31^35 channel Although we have not tested the sequence of permeability for di¡erent cations such as Na , K , and Ca2 , it is evident that ALP31^35 channels formed in membrane patches are cation-selective. The decrease of CsCl concentration in bath solution alters Er by 316.2 mV (Fig. 5), which is very close to the calculated Cs equilibrium potential of 317.8 mV. The PCs /PCl of 23 also indicates that the channel is more permeable for cations than for anions. It has been reported that the permeability sequence of ALP1^40 channel for di¡erent cations is PCs s PLi s PCa = PK s PNa in arti¢cial membrane (Arispe et al., 1993b). If ALP31^35 channels, as ALP1^ 40 channels, permit inward £ow of Ca2 preferentially, Ca2 overload would be the most serious consequence of the formation of new channels. Thus, the resultant disruption in Ca2 homeostasis would act as a key toxic factor of ALP leading to AD as proposed previously (Hardy and Higgins, 1992; Mattson et al., 1992; Wiss et al., 1994; Korotzer et al., 1995; Mattson, 1997; Ueda et al., 1997; Ma et al., 1998).
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Blockade of ALP31^35 channel activity by Zn2+ and Cd 2+
Fig. 7. Reversible blockade of channel formed by fragment 31^35 of ALP by Cd2 . Top panel showed the continuous channel activities induced by ALP31^35 added to the outer side of the patch. After addition of 1 mM CdCl2 to the inner side of the patch, obvious blockade of channel activities was observed (middle) ; the recovery of channel currents after replacing the Cd2 -containing bath solution with normal bath solution is shown in the bottom panel. Vp was 340 mV during all recordings.
Since Zn2 can bind to ALP molecules with high a¤nity (Bush et al., 1994a), it is probable that such Zn2 binding to ALP channels will induce functional alterations of ALP channel. Arispe et al. (1996) have observed that the conductance of ALP1^40 channel in planar bilayers is reduced by Zn2 in a dose-dependent manner. The results in the present experiment also demonstrate that Zn2 can block the channels formed by ALP31^35 in membrane patches of hippocampal neurons. Accordingly, the present data suggest that Zn2 could attenuate the neurotoxicity of ALP by blocking the channels formed by ALP or its fragments. The data also support the proposed clinical use of Zn2 supplementation in the treatment of AD (Potocnik et al., 1997). Zn2 also promotes the aggregation of ALP molecules and amyloid deposition (Bush et al., 1994b), and thereby it might contribute to the formation of senile plaques in AD. Therefore, the interaction of Zn2 with ALP molecule may be multiple, and the exact e¡ects and clinical signi¢cance of Zn2 on neurons in AD remain to be explored further. The blockade of ALP25^35 channels in arti¢cial lipid membrane by Cd2 has been reported (Mirzabekov et al., 1994). Our present experiments have also shown a suppressive e¡ect of Cd2 on ALP31^35 channels in neuronal membranes. The blockade of ALP31^35 channels by Cd2 implies the interaction of Cd2 with ALP31^35 molecules, and the subsequent modi¢cation of channel structure. In summary, our results demonstrate that ALP31^35 forms cation-selective, Zn2 - and Cd2 -sensitive ion channels in membrane patches excised from hippocampal neurons. It is suggested that fragment 31^35 is the shortest ALP active sequence known to date to form new ion channels across the cell membrane. This fact should be considered in the development of strategies for preventing the neurotoxic action of ALP in AD. AcknowledgementsöWe are grateful to Mr. Raymond Kong for his linguistic and grammatical contributions to this paper.
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Neuroscience Vol. 105, No. 4, pp. 845^852, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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AMYLOID L-PROTEIN FRAGMENT 31^35 FORMS ION CHANNELS IN MEMBRANE PATCHES EXCISED FROM RAT HIPPOCAMPAL NEURONS J.-S. QI and J.-T. QIAO* Department of Neurobiology, Shanxi Medical University, Taiyuan, Shanxi 030001, PR China
AbstractöInside-out membrane patches excised from rat hippocampal neurons were used to test if ion channels could be formed by fragment 31^35 of amyloid L-protein. The results showed: (1) after application of fragment 31^35 of amyloid L-protein (5 WM) to either the inner or outer side of the patches, spontaneous currents could be recorded from those patches that had previously been `silent'; (2) the fragment 31^35-induced conductance was cation-selective with a permeability ratio of PCs /PCl = 23; (3) di¡erent levels of conductance, ranging from 25 to 500 pS, could be recorded in di¡erent patches, and in some cases, di¡erent conductances and spontaneous transitions among them could be recorded in a single patch; and (4) application of ZnCl2 (1 mM) to the inner side of the patches reversibly blocked the newly formed channel activity; a similar e¡ect was observed after application of CdCl2 (1 mM). These results show that fragment 31^35 of amyloid L-protein can insert into membrane patches from both sides and form cation-selective, Zn2 - and Cd2 -sensitive ion channels. It is proposed that fragment 31^35 in amyloid L-protein might be the shortest active sequence known to date to form ion channels across neuronal membranes. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: amyloid L-protein fragment 31^35, inside-out membrane patch, hippocampal neurons, formation of ion channels, Zn2 , Cd2 .
excised from hypothalamic neurons. Simmons and Schneider (1993), using the whole cell voltage clamp technique, have reported that ALP25^35, a short form of ALP fragments which is also neurotoxic, induces a novel ionic conductance in sympathetic ganglion neurons. Furthermore, the observation of Mirzabekov et al. (1994) has shown that ALP25^35 spontaneously incorporates into planar lipid bilayers to form ion-permeable channels. We have demonstrated in a recent study (Yan et al., 1999) that ALP31^35, a shorter fragment of ALP, is able to induce apoptosis in cultured cortical neurons similar to that observed with ALP1^40 or ALP25^35. The neurotoxic action of ALP31^35 has also been shown in cultured basal forebrain GABAergic neurons (Pakaski et al., 1998) and septal cholinergic neurons (Wang et al., 1999). Here, we have investigated whether this shorter fragment of ALP, ALP31^35, can form ion channels across the membrane patch of acutely dissociated hippocampal neurons.
Several lines of evidence have demonstrated that amyloid L-protein (ALP), a main component of the senile plaques in Alzheimer's disease (AD), is neurotoxic and contributes to neuronal dysfunction and death (Pike et al., 1991; Blanchard et al., 1997; Mattson, 1997). The mechanisms of ALP neurotoxicity may involve calcium overloading (Wiss et al., 1994; Korotzer et al., 1995; Ueda et al., 1997; Ma et al., 1998), generation of free radicals (Behl et al., 1994; Manelli and Puttfarcken, 1995; Goodman et al., 1996), modulation of endogenous channels (Etcheberrigaray et al., 1994; Fraser et al., 1997), and/or the formation of new ion channels (Arispe et al., 1993a,b; Mirzabekov et al., 1994; Kawahara et al., 1997; Lin et al., 1999). Among the neurotoxic e¡ects cited above, the formation of new ion channels has provoked extensive interests. Arispe et al. (1993a,b) and Kawahara et al. (1997) have shown that ALP1^40, the commonly observed type of ALP in senile plaques, can form new cation channels in arti¢cial lipid bilayers and in membrane patches
EXPERIMENTAL PROCEDURES
Solutions and isolation of hippocampal neurons
*Corresponding author. Tel.: +86-0351-4135052; fax: +86-03514083014. E-mail address: [email protected] (J.-T. Qiao). Abbreviations : ALP, amyloid L-protein; ALP31^35, fragment 31^35 of amyloid L-protein ; ALP25^35, fragment 25^35 of amyloid Lprotein; ACSF, arti¢cial cerebrospinal £uid; AD, Alzheimer's disease; EGTA, ethylene glycol-bis(L-aminoethyl ether)N,N,NP,NP-tetraacetic acid; Er , reversal potential ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulfonic acid); Po , probability of channel opening; Vp , potential in pipette.
The following solutions (in mM) were used during the course of the dissociation procedure: (1) arti¢cial cerebrospinal £uid (ACSF): NaCl 126, KCl 5, CaCl2 2, MgSO4 2, NaH2 PO4 1.5, NaHCO3 25, glucose 10 (pH 7.4); (2) ACSF with lower concentration of calcium: NaCl 126, KCl 5, CaCl2 0.2, MgSO4 2, NaH2 PO4 1.5, NaHCO3 25, glucose 10 (pH 7.4); and (3) incubation solution: NaCl 130, KCl 5.4, CaCl2 1, MgCl2 1, HEPES 10, glucose 25 (pH 7.4). Symmetrical CsCl solution, that was used as both bath and pipette solution during single channel 845
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recording, possessed the following components (mM): CsCl 140, CaCl2 0.5, MgCl2 0.5, EGTA 1, HEPES 5 (pH 7.4). The experiments were carried out on 28 Wistar rats aged about 20 days (supplied by the Research Animal Center of Shanxi, PR China, with approval of Shanxi Committee on Ethics of Animal Research), and all e¡orts have been made to minimize the number of animals used and their su¡ering. Single neurons were dissociated from the hippocampus as described by Kay and Wong (1986) with minor modi¢cations. In brief, the brain was rapidly removed under the anesthesia with ether and placed in cold (0V3³C) ACSF with a lower concentration of calcium; the hippocampus was dissected out from the cooled brain tissue; slices of 400^600 Wm thickness were cut with a razor blade and transferred into the ACSF bubbled with 95% O2 and 5% CO2 . After at least 1 h, the hippocampal CA1 regions were micro-dissected and cut into small pieces of about 1 mm2 in incubation solution; then the pieces were placed into the incubation solution containing pronase E (1 mg/ml, Merck, Germany) and treated for 30 min at 33³C. After digestion, the pieces were dispersed by trituation with a graded series of Pasteur pipettes. Then, three drops of cell suspension were added to coverslip coated with poly L-lysine (0.1%) to improve cell adherence. After cells settled down on the glass bottom, the incubation solution was replaced with 140 mM CsCl solution (recording solution) before sealing the patch pipette.
ALP channel current recording and single channel analysis Patch clamp experiments were performed following standard procedures (Hamill et al., 1981). Recording pipettes were made on a two-step vertical puller (PP-830, Narishige), and ¢re-polished with a microforge (MF-830, Narishige). The pipette resistance, when ¢lled with 140 mM CsCl solution, was between 8 and V15 M6. Inside-out con¢guration of membrane patch was obtained by rapidly withdrawing the pipette after forming a giga-ohm seal. Channel currents were ¢ltered at 1 or 2 kHz (33 dB), ampli¢ed by a patch clamp ampli¢er (Axopatch 200B, Axon Instruments, USA), and stored on the hard disk of an IBM computer through a 12-bit 333 kHz A/D converter (Digidata 1200B, Axon Instruments, USA). Single channel data acquisition and o¡-line analysis were carried out with PCLAMP 6.04 software (Axon Instruments, USA). Administration of ALP fragments ALP31^35 and ALP25^35 were dissolved in double distilled water as concentrated stock solutions (1 mM) and stored at 320³C. The ¢nal concentration of ALP fragments added to bath solution or pipette solution was 5 WM. Before adding ALP fragments to bath solution (i.e. inner or cytoplasm side of the patch), the excised membrane patch was exposed to sym-
Fig. 1. Representative channel activities induced by adding fragment 31^35 of ALP (5 WM) to the outer side of an inside-out membrane patch. Recordings shown here were obtained 6 min after application of ALP31^35 in symmetrical CsCl solution (140 mM). Vp is indicated above each set of tracings. Channel openings are upward when Vp is negative, or downward when Vp is positive.
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metrical Cs solution for at least 10 min in order to verify that the membrane patch was electrically `silent' by blocking the possibly existed endogenous channels in the patch, as mentioned below. After appearance of ALP channel activity, bath solution was replaced with CsCl solution containing 1 mM ZnCl2 or 1 mM CdCl2 through a gravity-driven multitube perfusion system. The distance from the opening of perfusion tube to the tip of patch pipette was about 100 Wm. In the case of application of ALP fragments to pipette solution (i.e. outer or extracellular side of the patch), the tip of pipette was ¢rst back-¢lled with CsCl solution, then ALP fragments solution was added to the distal end of the CsCl solution in the pipette. Thereby, when it was used for recording, a control period in the absence of ALP was achieved (Kawahara et al., 1997). ALP31^35, ALP25^35, CsCl, ZnCl2 , and CdCl2 were all purchased from Sigma (St. Louis, MO, USA). Experiments were performed at room temperature (22^27³C). The statistical data are expressed as means þ S.D. A P value of = 0.05 was considered statistically signi¢cant.
RESULTS
Endogenous potassium channels in the excised patches were blocked by using a K -free solution containing 140 mM CsCl on both sides of membrane patches. Calcium current was prevented by adding 1 mM EGTA and sodium channels were inactivated by holding the membrane potential between 340 and V+40 mV. To ensure the absence of endogenous channels, recordings were made from a control group with no application of ALP fragments in the bath or pipette solutions. For each patch, an initial control recording was made at a high ampli¢er gain (100 mV/pA) and with di¡erent levels of potential in pipette (Vp , 340V+40 mV) before application of ALP fragments to ensure the absence of endogenous activity. The minimum control time was 10 min or 4 min in experiments for bath or pipette application of ALP fragments, respectively. ALP31^35 forms ion channels across neuronal membrane patches similar to ALP25^35 Under the above conditions, most of the patches that
Fig. 2. Open-time distribution of channel currents induced by fragment 31^35 of ALP. Data were obtained at +40 mV of Vp and symmetric CsCl solution ; ALP31^35 (5 WM) was added to the outer side of the patch. The open-time histogram was best ¢tted by a two-exponential function. The time constants d1 , and d2 are indicated in the graph.
Fig. 3. Di¡erent amplitude of channel currents induced by fragment 31^35 of ALP at the same Vp (340 mV). Symmetric CsCl solutions were used, and ALP31^35 (5 WM) was added to the outer side of the patch. The four groups of recordings (A, B, C and D) came from di¡erent membrane patches. Main conductance values estimated were 34, 55, 82, and 328 pS in A, B, C, and D, respectively. Note that di¡erent levels of conductance were also observed in the same membrane patch (B and C).
were untreated with ALP fragments (10/11) remained electrically silent for up to 60 min. Only 9% (1/11) of these patches displayed occasional current activities. However, channel activities appeared in all patches (100%, 7/7) between 4 and V16 min after exposure of the inner side of the membrane patches to 5 WM ALP25^ 35; 93.5% (14/15) of patches displayed similar channel currents when ALP25^35 was added to the outer side of the membrane via the pipette. A shorter fragment, ALP31^35, composed of only ¢ve amino acids, induced similar channel activities in 92.3% (12/13) or 87.5% (7/8) of membrane patches after the peptide was added into bath or pipette solution, respectively. Fig. 1 shows the single channel activity induced by adding ALP31^35 to the outer side of a patch at di¡erent Vp . The histogram of ALP31^35 current amplitudes could be ¢tted well to a normal distribution (data not shown); the mean open time (Topen ) for the channels was 1.20 þ 0.31 ms (n = 8) at +40 mV of Vp . An open-time histogram was best ¢tted by a two-exponential function (Fig. 2). Characteristics of ALP fragment-induced channel activity Multilevel conductance and spontaneous transition. Unlike the endogenous channels, ALP fragment-induced currents in most of patches showed substantial variability in amplitude. The conductances of the ALP31^35-
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Fig. 4. Spontaneous transition between the di¡erent levels of conductance formed by adding fragment 31^35 of ALP. Graphs A, B, C, and D represent sample currents from four di¡erent membrane patches. Symmetrical CsCl solution was used. Vp was given below in each recording. Numbers on the right side of each recording represent the di¡erent conductance values, and `c' represents closed state of the channels.
induced channels ranged from 25 to 500 pS (n = 19). Fig. 3 illustrates the variability in amplitudes of wellde¢ned channel currents from four di¡erent membrane patches; the main values of conductance from samples A^D, respectively, were 34, 55, 82, and 328 pS at the same Vp (340 mV). In addition, the spontaneous transitions of conductance between di¡erent levels were also found in the same patches, though it occurred in not more than one-¢fth of patches with well-de¢ned channel currents. These spontaneous transitions are illustrated in Fig. 4. Transitions of two sample currents from lower conductance to higher conductance are depicted in A and B, being increased by 80% and 83%, respectively; sample C shows frequent transitions of a channel conductance among di¡erent levels; while the records in D show abrupt changes in conductance from 78 pS to 150 pS and from 115 pS to 195 pS, respectively. The fact that the channel could be closed abruptly and completely from the higher level of conductance excludes the possibility that the existence of a higher level of conductance is owing to the `summation' of conductance from two or more channels. The characteristics of multilevel conductance and spontaneous transition were also observed when ALP25^35 was used (data not shown). Voltage dependence of channel opening. The open frequency of the channels changed with the alteration of transmembrane potential. Topen and the probability of channel opening (Po ) showed similar voltage dependency. For example, when Vp was changed from +20 mV to +40 mV, Topen increased from 0.842 þ 0.27 ms to 1.203 þ 0.31 ms (n = 8, P 6 0.05), and the mean Po from 0.018 þ 0.011 to 0.07 þ 0.05 (n = 6, P 6 0.05). Cation-selectivity of ALP31^35 channel. To identify the ion carrying the current, we observed the channel activities induced by ALP31^35 and measured the shift of the reversal potential (Er ) while changing the [Cs ]i
from 140 mM to 70 mM (n = 14). As expected, the channel current was about 0 pA in symmetrical CsCl solution ([Cs ]i /[Cs ]o = 140/140 mM) at 0 mV of Vp ; by contrast, when asymmetrical CsCl solution ([Cs ]i /[Cs ]o = 70/140 mM) was used, obvious inward currents were invariably observed at Vp = 0 mV. Furthermore, in the experiments for measuring the Er , it was found that Er , being about 0 mV in symmetrical solution, was shifted towards the negative value of Vp in asymmetrical solution, becoming 316.2 mV (Fig. 5). The value of the new Er is close to Cs equilibrium potential (317.8 mV), and it corresponds to a PCs /PCl of 23. This result indicated that under these conditions, the main charge carrier of current is Cs . Blockade of ALP fragment-induced channel activity by Zn2+ and Cd 2+ Representative experiments in Fig. 6 show that addition of ZnCl2 (1 mM) to the bath solution drastically reduced the amplitude and the open frequency of channel currents induced by ALP31^35 (Fig. 6A) or ALP25^ 35 (Fig. 6B) applied through pipette solution. This action seemed to be reversible, in that the channel activity reappeared when Zn2 -containing bath solution was replaced with normal bath solution. We also examined the e¡ect of Cd2 on the channel activity induced by ALP fragments (n = 9). CdCl2 (1 mM) blocked ALP31^35-induced channel currents in 8/9 patches tested (Fig. 7). The Po of the channel in CdCl2 decreased from 0.035 to 0.021, and the average current amplitude from 3.17 þ 0.632 to 1.324 þ 0.38 pA. The suppressive action of Cd2 on ALP31^35 channels was reversible.
DISCUSSION
Since Arispe et al. (1993a,b) found that ALP1^40
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Fig. 5. Channels formed by fragment 31^35 of ALP are cation-selective. (A) Channel activities recorded in symmetrical and asymmetrical CsCl solution. Di¡erent levels of Vp are shown on the left of corresponding recordings. The solid line in each trace represents channel closed state. (B) Amplitudes of ALP31^35 channel currents plotted as a function of Vp . Er was shifted from about 0 mV in control (b) to 316.2 mV after asymmetrical CsCl solution was used (a). PCs /PCl was estimated using the following equation: Er = (RT/F) ln{{PCs [Cs]o +PCl [Cl]i }/{PCs [Cs]i +PCl [Cl]o }}, by inserting the values of concentrations that were presently used ([Cs]o = 140 mM, [Cs]i = 70 mM, [Cl]o = 140 mM, [Cl]i = 70 mM) into the equation, and the result showed: PCs /PCl = 23.
could induce a novel inward ionic conductance in bilayer membranes, the mechanisms by which ALP a¡ects ion £ux across the membrane have been widely studied. The present results demonstrate that not only ALP25^35 but also ALP31^35 can spontaneously insert into membrane patches from both sides and form cation-selective, Zn2 and Cd2 -sensitive ion channels. The characteristics of
ALP31^35 channels are quite similar to that of channels formed by ALP1^40 or ALP25^35 in arti¢cial lipid bilayers (Arispe et al., 1993a,b; Mirzabekov et al., 1994) or neuronal membrane (Simmons and Schneider, 1993; Kawahara et al., 1997) as reported previously. ALP31^35 is the shortest sequence of ALP known to date to form transmembrane channels.
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Fig. 6. Reversible blockade of channels formed by fragments 31^35 and 25^35 of ALP by Zn2 . Membrane patches were exposed to symmetrical CsCl solutions, and ALP31^35 (A) or ALP25^35 (B) was applied from the outer side of the patch. After appearance of channel currents (top), bath solution containing 1 mM of ZnCl2 was added to the inner side of the patches (middle); the channel activities in recovery were recorded 2 min later after replacing Zn2 -containing bath solution with normal bath solution (bottom). Vp was 340 mV during all recordings above.
Formation of ALP31^35 channel An important question is how a small peptide of only ¢ve amino acids might form a membrane-spanning ion channel. It has been reported (Barrow et al., 1992) that ALP and some of its degradation molecules, in aqueous solution, can form stable dimers, trimers and tetramers and, moreover, the rate of aggregation is the fastest for the hydrophobic sequence 29^42 of ALP. In addition, the critical role of isoleucine in 31 and 32 and of methionine in 35 position for the aggregation and toxicity of ALP25^ 35 has been emphasized (Pike et al., 1993). Considering the facts that ALP fragment 31^35 (Ile-Ile-Gly-Leu-Met) is constituted of hydrophobic amino acids and contains all of the three key amino acids responsible for the aggregation and toxicity of ALP25^35, we postulate that this shorter peptide cannot only exhibit the apoptogenic activity on neurons, as proved previously (Yan et al., 1999), but also can easily incorporate itself into the membrane and form special membrane-spanning ionophore structures. As a corollary, if new channels could be formed by the aggregation of ALP31^35 fragments, different polymers incorporated into the cell membrane would be responsible for the di¡erent levels of conductance; the spontaneous transition of conductance states, as observed in the present study, might be the result of the rapid inter-conversion among di¡erent conforma-
tions of the same ALP31^35 polymers. As pointed out by Durell et al. (1994), the inter-conversion does exist among di¡erent subtypes of polymers formed by ALP fragments. Cation-selectivity of ALP31^35 channel Although we have not tested the sequence of permeability for di¡erent cations such as Na , K , and Ca2 , it is evident that ALP31^35 channels formed in membrane patches are cation-selective. The decrease of CsCl concentration in bath solution alters Er by 316.2 mV (Fig. 5), which is very close to the calculated Cs equilibrium potential of 317.8 mV. The PCs /PCl of 23 also indicates that the channel is more permeable for cations than for anions. It has been reported that the permeability sequence of ALP1^40 channel for di¡erent cations is PCs s PLi s PCa = PK s PNa in arti¢cial membrane (Arispe et al., 1993b). If ALP31^35 channels, as ALP1^ 40 channels, permit inward £ow of Ca2 preferentially, Ca2 overload would be the most serious consequence of the formation of new channels. Thus, the resultant disruption in Ca2 homeostasis would act as a key toxic factor of ALP leading to AD as proposed previously (Hardy and Higgins, 1992; Mattson et al., 1992; Wiss et al., 1994; Korotzer et al., 1995; Mattson, 1997; Ueda et al., 1997; Ma et al., 1998).
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Blockade of ALP31^35 channel activity by Zn2+ and Cd 2+
Fig. 7. Reversible blockade of channel formed by fragment 31^35 of ALP by Cd2 . Top panel showed the continuous channel activities induced by ALP31^35 added to the outer side of the patch. After addition of 1 mM CdCl2 to the inner side of the patch, obvious blockade of channel activities was observed (middle) ; the recovery of channel currents after replacing the Cd2 -containing bath solution with normal bath solution is shown in the bottom panel. Vp was 340 mV during all recordings.
Since Zn2 can bind to ALP molecules with high a¤nity (Bush et al., 1994a), it is probable that such Zn2 binding to ALP channels will induce functional alterations of ALP channel. Arispe et al. (1996) have observed that the conductance of ALP1^40 channel in planar bilayers is reduced by Zn2 in a dose-dependent manner. The results in the present experiment also demonstrate that Zn2 can block the channels formed by ALP31^35 in membrane patches of hippocampal neurons. Accordingly, the present data suggest that Zn2 could attenuate the neurotoxicity of ALP by blocking the channels formed by ALP or its fragments. The data also support the proposed clinical use of Zn2 supplementation in the treatment of AD (Potocnik et al., 1997). Zn2 also promotes the aggregation of ALP molecules and amyloid deposition (Bush et al., 1994b), and thereby it might contribute to the formation of senile plaques in AD. Therefore, the interaction of Zn2 with ALP molecule may be multiple, and the exact e¡ects and clinical signi¢cance of Zn2 on neurons in AD remain to be explored further. The blockade of ALP25^35 channels in arti¢cial lipid membrane by Cd2 has been reported (Mirzabekov et al., 1994). Our present experiments have also shown a suppressive e¡ect of Cd2 on ALP31^35 channels in neuronal membranes. The blockade of ALP31^35 channels by Cd2 implies the interaction of Cd2 with ALP31^35 molecules, and the subsequent modi¢cation of channel structure. In summary, our results demonstrate that ALP31^35 forms cation-selective, Zn2 - and Cd2 -sensitive ion channels in membrane patches excised from hippocampal neurons. It is suggested that fragment 31^35 is the shortest ALP active sequence known to date to form new ion channels across the cell membrane. This fact should be considered in the development of strategies for preventing the neurotoxic action of ALP in AD. AcknowledgementsöWe are grateful to Mr. Raymond Kong for his linguistic and grammatical contributions to this paper.
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